Extracorporeal medical machines such as the pump-oxygenator and artificial kidney rely on systemic heparinization to improve blood compatibility. However, heparin can lead to serious complications such as bleeding. With the prospect of even longer perfusion times with machines such as the membrane oxygenator, the problems due to heparinization become more severe. Although many approaches have been explored to solve this problem, such as the use of neutralizing compounds or heparin bonded surfaces, there still remains no real alternative to systemic heparinization. We propose a new method to control heparin levels using a blood filter containing immobilized heparinase. Such a filter might be used in situations where it is desired to heparinize the extracorporeal circuit without simultaneous heparinization of the patient. Alternatively, it could eliminate the use of neutralizing compounds such as protamine. Because of the amount of data on heparinase has until now been limited and the methods of producing it are inadequate for large scale use, the principal focus of our research during the two years and one month of our initial grant has been on conducting feasibility studies on heparinase production, purification, and immobilization. The principal findings of our work thus far include 1) increasing volumetric enzyme production over 1,000-fold from previous published procedures, 2) purifying heparinase by over 1,000-fold from the crude cell extracts, 3) characterizing the biochemical properties of heparinase, 4) isolating the first heparinase inhibitors, 5) immobilizing heparinase with 91% activity recovery and excellent stability, and 6) demonstrating that columns as small as 1.5 ml can remove clinically used quantities of heparin at low flow rates in aqueous medium and in blood. The specific objectives of this proposal are to (1) optimize enzyme production and completely purify heparinase, (2) develop a biocompatible immobilized heparinase filter and test it in vitro, and (3) conduct animal tests with this filter.